In addition to mediating activation of CD4 T cells, antigenic peptide-MHC class II complexes transduce signals leading to antigen presenting cell activation. Using well-characterized anti-I-Ak monoclonal antibodies (mAbs) that have been available for over 20 years, we recently established that these mAbs differ in their ability to elicit class II signaling in resting B cells. Specifically, a mAb that binds the 1 chain Ia.2 epitope elicits src family kinase-mediated calcium signaling, while two mAbs that bind the 2 chain Ia.17 epitope do not. In preliminary data presented in this proposal, we further demonstrate that the Ia.2 epitope is not present on all I-Ak molecules as previously thought, but rather marks a subset of cell surface I-Ak class II molecules. Further, in addition to its unique signaling capacity the Ia.2+ subset of I-Ak molecules is highly enriched in lipid rafts, critically involved in initiation of cognate MHC-restricted B cell-T cell interactions, and uniquely associated with a low MW cell surface protein. Based on these published and preliminary results, we put forth the hypothesis that the Ia.2 epitope represents a unique molecular structure generated, or rendered accessible, on a subset of signaling-competent lipid raft-tropic I-Ak class II molecules, which are critically important to key MHC class II functions such as B cell signaling and formation of cognate B cell-T cell interactions. To test this hypothesis, we will accomplish two Specific Aims. First, we will further establish the molecular structure of the Ia.2-bearing lipid raft-tropic signaling-competent subset of I-Ak class II molecules. Second, we will establish the sub-cellular compartment in which Ia.2- bearing I-Ak class II molecules are formed. Completion of these specific aims will reveal the basic molecular mechanism behind the unique signaling properties of Ia.2+ I-Ak class II molecules, establish a molecular mechanism controlling the selective lipid raft partitioning of Ia.2+ I-Ak class II molecules (and other MHC class II haplotypes), and lay the groundwork for the further characterization the role Ia.2+ and other MHC class II subsets in BCR-mediated antigen processing / presentation. PUBLIC HEALTH RELEVANCE: B cell and other antigen presenting cells (APCs) express cell surface proteins called major histocompatibility complex (MHC) class II molecules. Cellular activation signals delivered to these cells through MHC class II molecules are important physiologically for antibody-based immunity (e.g. successful vaccination). Therapeutically, anti-MHC II monoclonal antibodies have been used in lymphoma patients to help eliminate malignant cells by triggering cell death. The increased knowledge of B cell MHC class II signaling gained during this research project will support a greater ability to harness MHC class II signaling for improved immunization and anti-tumor therapy.